Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-based HIV-1 genome editing has shown promising outcomes in in vitro and in vivo viral infection models. However, existing HIV-1 sequence variants have been shown to reduce CRISPR-mediated efficiency and induce viral escape. Two metrics, global patient coverage and global subtype coverage, were used to identify guide RNA (gRNA) sequences that account for this viral diversity from the perspectives of cross-patient and cross-subtype gRNA design, respectively. Computational evaluation using these parameters and over 3.6 million possible 20-bp sequences resulted in nine lead gRNAs, two of which were previously published. This analysis revealed the benefit and necessity of considering all sequence variants for gRNA design. Of the other seven identified novel gRNAs, two were of note as they targeted interesting functional regions. One was a gRNA predicted to induce structural disruption in the nucleocapsid binding site (Ψ), which holds the potential to stop HIV-1 replication during the viral genome packaging process. The other was a reverse transcriptase (RT)-targeting gRNA that was predicted to cleave the subdomain responsible for dNTP incorporation. CRISPR-mediated sequence edits were predicted to occur on critical residues where HIV-1 has been shown to develop resistance against antiretroviral therapy (ART), which may provide additional evolutionary pressure at the DNA level. Given these observations, consideration of broad-spectrum gRNAs and cross-subtype diversity for gRNA design is not only required for the development of generalizable CRISPR-based HIV-1 therapy, but also helps identify optimal target sites.

Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection

“…The strategies that target viral sequences, such as HIV DNA excision, may encounter problems with HIV diversity and the selection of effective sgRNAs ( Yoder, 2019 ). In such cases, a personalized approach in sgRNA design would help make therapy more effective ( Dampier et al., 2018 ; Chung et al., 2021 ).…”

How to break free: HIV-1 escapes from innovative therapeutic approaches

“…They compared single and dual gRNAs targeting different HIV-1 regions and found that the latter approach showed no virus replication and no escape for up to 60 days, extended to an even longer period for gRNA combinations targeting highly conserved sites (112). To aid the design of more broad-acting and effective gRNA sequences, in silico predictive pipelines based on sequences shared by genetic variants and viral subtypes have been developed (113,114). This further optimization may help in reducing viral escape from the CRISPR-Cas9 system.…”

Targeting and Understanding HIV Latency: The CRISPR System against the Provirus

“…Furthermore, the problem of the emergence of resistant viruses may be overcome by targeting highly conserved sequences, as it is less probable that a functional resistant mutant could be generated. It is well known that HIV-1 displays a great inter-and intra-patient variability, so bioinformatic pipelines have come in handy to predict what are the most conserved sequences among different variants and, thus, select the possible most efficient broad-spectrum gRNAs in order to be effective in as many patients as possible [42,43].…”